The present invention is directed to a method of increasing the effectiveness of molecules that are phosphorylated in their active state. This is accomplished by transducing cells with the gene for deoxycytidine kinase resulting in the chemosensitization of such cells which are targets for those molecules. Preferably, the target cells are virally infected cells and/or tumor cells. Preferred tumor cells are solid tumor cells such as brain tumors.
Deoxycytidine kinase (dCK) is an enzyme that catalyses the phosphorylation of a variety of pyrimidine and purine deoxynucleosides to their corresponding nucleotide [Ruiz van Haperen and Peters, Urine and Pyrimidine Metabolism 15: 104-112 (1994)]
A number of the above-mentioned deoxynucleoside molecules when phosphorylated by dCK are xe2x80x9cactivatedxe2x80x9d and display an antineoplastic and/or antiviral activity. For example Ara-C (1-xcex2-D-arabinofuranosyl-cytosine also referred to as cytarabine) is presently one of the most effective agents in the treatment of acute myeloid leukemia [Ellison, R. R., et al. Blood 32:407-523 (1968); Cadman, E. et al., Cancer Treat. Rep. 61:1109-1116 (1977); Balwell, B., et al., Leukemia 2: 253-60 (1988); Momparler, R. L., et al. Drug Resistance to Cytosine Arabinoside, in: Kessel D., Ed, Resistance to Antineoplastic Drugs, Boca Raton; CRC Press, 353-67 (1989); Grant, S., Pharmacol. Ther. 48:29-44 (1990)]. Ara-C is incorporated into replicating DNA and terminates DNA chain elongation [Graham, F. L., et al., Cancer Res. 30:2636-3644 (1970); Kufe, W. D., et al., J. Biol. Chem. 225:8997-9000 (1980); Kufe, D., et al., Blood 64:54-58 (1984)]. Unfortunately Ara-C has shown limited activity against most solid tumors including brain tumors. Aza-CdR (sometimes referred to as decitabine or 5-aza-2xe2x80x2-deoxycytidine) has shown considerable activity in treating both experimental leukemia and human acute myelogenous leukemia [Richei, D., et al., Br. J. Cancer, 58:730-3 (1988) Richei, D., et al., Contrib. Oncology, 37:20-9 (1989) Pinto, A., et al., Leukemia Supp,. 1:51-60 (1993)]. dFdC (2,2xe2x80x2-difluorodeoxycytidine or gemcitabine) [Heinemann, V., et al., Cancer Res. 48:4024-31 (1988)] has shown preclinical activity against a number of experimental tumors and human xenografts [Hertel, L. W., et al., Cancer Res. 50:4417-4422 (1990); Braakhuis, B. J. M., et al., Cancer Res., 51:211-214 (1991); Boven E., et al., Br. J. Cancer 68:52-56 (1993)]. Other molecules include cladribine (2-chloro-2xe2x80x2-deoxyadenosine) [Erikson, S., et al., Biochem. Biophys. Res. Commun., 176:586-592 (1991)], zalcitabine (2xe2x80x2, 3xe2x80x2-dideoxycitidine) [Datta, N. S., et al., Biochemistry 28:114-123 (1989); Sarup, J. C., et al., Biochem. Biophys. Res. Commun., 176:586-592 (199 1)] and fludarabine (9-xcex2-D-arabinofuranosyl-2-fluoroadenine) [Tseng W., et al., Mol. Pharmacol. 21:474-477 (1982)].
The mode of activation of these molecules is similar. For example, fludarabine monophosphate is dephosphorylated extracellularly to fludarabine, which is then transported into the cell. Where dCK phosphorylates this molecule [Verhoef, V., et al. Cancer Res. 41:4478-4483 (1981)] into the monophosphate form which is then converted to the triphosphate derivative, which is a potent inhibitor of DNA polymerase and ribonucleotide reductase.
dCK phosphorylates cladribine into its active state [Carson, D. A., et al., Proc. Natl. Acad. Sci. USA, 77:6865-6869 (1980)], where it exhibits activity against a number of leukemia cell lines, but has not shown much activity against most non-lymphoid solid tumors [Carson, D. A., et al., Blood 62:737 (1983)]. However, it has been found to be active against the human ovarian carcinoma cell line A2780 [Ruiz van Haperen, V. W. T., et al., Proc. Am. Assoc. Cancer Res., 34:307 (1993)].
It would be very valuable if the relative effectiveness of any of these molecules could be increased. This could be accomplished, for example, by permitting the administration of lower dosages of these molecules or by extending the range of tumors or viruses that these molecules can be used against.
The effectiveness of some chemotherapeutic agents has been enhanced by increasing the sensitivity of cells to such agents. For example, transduction of tumor cells by the herpes simplex thymidine kinase (hstk) gene has sensitized certain cells to agents which are not otherwise effective. Hstk phosphorylates, and thereby activates, nucleoside analogs such as ganciclovir (GCV) and acyclovir (ACV) [Moolten, F. L., Cancer Res. 46:5276-5281 (1986)]. Normal eukaryotic cells fail to phosphorylate GCV and are therefore relatively resistant to the drug. In contrast, those cells transduced with hstk convert GCV/ACV to the lethal phosphorylated intermediate. The identification of other chemosensitization gene/prodrug systems would be very valuable.
We have now identified a new method for enhancing the effectiveness of a group of molecules that are phosphorylated or capable of phosphorylation by dCK. Thus, we have identified a new chemosensitization xe2x80x9cgene/prodrugxe2x80x9d system. This system involves using dCK as the gene and molecules activated by dCK phosphorylation as the prodrug. The molecules that can be used are those that can be used against leukemia cells. These molecules include ara-C [Durham, J. P., et al., Mol Pharmacol., 5:358-375 (1969); Ho. D. H. W., et al., Cancer Res. 33:2816-20 (1973); Habteyesus, A., et al., Biochem. Pharmacol. 42:1829-1836 (1991); Datta, N. S., et al., Biochemistry 28:114-123 (1989); Durham, J. P., et al., J. Biol. Chem., 245:2276-2284 (1970); Erickson, S., et al., Biochem. Biophys. Res. Commun., 176:586-592 (1991)], dFdC [Heinemann, V., et al., Cancer Res., 48:4024-4031 (1988)], cladribine [Sarup, J. C., et al., Biochem. Pharmacol. 38:2601-2607 (1989)], zalcitabine [Datta, N. S., et al., Biochemistry 28:114-123 (1989); Srup, J. C., et al., Biochem. Pharmacol., 38:2601-2607 (1989); Erickson, S., et al., Biochem. Biophys. Res. Commun., 176:586-592 (1991)], and fludarabine [Tseng W., et al., Mol Pharmacol., 21:474-477 (1982)]. Phosphorylization of these molecules yields the corresponding nucleoside triphosphate which exhibits an antiviral, antineoplastic, etc. activity.
One preferred way of increasing the effectiveness of these molecules is by increasing the sensitization of the target cells to these molecules. That can be accomplished by increasing the levels of dCK expressed. We have discovered that one way of accomplishing this is by introducing a dCK gene into a cell, e.g. by transducing a target cell with a gene encoding dCK, preferably the human dCK gene. Nucleic acid delivery systems include viral vectors, catheters, chemical conjugates, and fusion proteins having (1) a targeting moiety such as an antibody specific for a target cell and (2) a nucleic acid binding moiety such as a protamine. Preferably the dCK gene is operably linked to a promoter and more preferably also includes an element that results in a high level of expression. Preferably the gene is under the control of an inducible promoter.